3 Advantages of detecting mutations with next-generation sequencingHigh throughputTest many genes at onceSystematic, unbiased mutation detectionAll mutation typesSingle nucleotide variants (SNV), copy number alteration (CNA)-insertions, deletions and translocationsDigital readout of mutation frequencyEasier to detect and quantify mutations in a heterogeneous sampleCost effective precision medicine“Right drug at right dose to the right patient at the right time”

14 Getting started Capture efficiency and coverageOverall and by geneSpecimen type differencesFresh-frozen vs. FFPE specimensDetection of single nucleotide variants (SNVs)MethodsFiltersDetection of indels and other mutation types

22 Defining clinical validationAccuracyDegree of agreement between the nucleic acid sequences derived from the assay and a reference sequencePrecisionRepeatability—degree to which the same sequence is derived in sequencing multiple reference samples, many times. Reproducibility—degree to which the same sequence is derived when sequencing is performed by multiple operators and by more than one instrument.Analytical SensitivityThe likelihood that the assay will detect a sequence variation, if present, in the targeted genomic region.Analytical SpecificityThe probability that the assay will not detect a sequence variation, if none are present, in the targeted genomic region.Diagnostic SpecificityThe probability that the assay will not detect a clinically relevant sequence variation, if none are present, in the targeted genomic region.

23 Reproducibility Test Type Definitions Inter-Tech (Stringent)The technicians performing the run were different, but the experiment and lanes were the same.Inter-Tech (Relaxed)The technicians performing the run were different for each comparison. We did not control for the experiment or lane.Intra-TechThe technician performing the run was the same. The experiment was different.Inter-Lane (All)The lanes are different. These experiments, the techs were different in two, and the same in two.Inter-Lane & Intra-TechThe lanes are different. In these experiments, the techs were the same.Intra-Lane & Inter-TechThe lanes are the same. In these experiments, the techs were different.

43 ConclusionsCancer NGS gene panel helps in multiplexing key actionable genes for a cost effective, accurate and sensitive assayTargeted cancer panel are useful for “drug repurposing” of small molecule inhibitorsClinical validation of NGS assays in cancer is complex and labor intensive but basic principles remainBioinformatic barriers are the most challenging